A method of using proteins of the SIRT family or of polypeptide or peptide fragments of SIRT proteins as an active ingredient for slimming, alone or in combination with at least one other active agent, in a cosmetic composition or for the preparation of a pharmaceutical and/or dermatological composition is described. Said peptides are intended, in particular, for treatment of cellulite and/or used to decrease, eliminate or prevent excess fat beneath the skin.”

The invention concerns the field of cosmetics and pharmaceuticals, in particular, the field of dermatology.
The purpose of the present invention is the utilization of proteins of the SIRT family or polypeptide or peptide fragments of SIRT proteins as an active ingredient for slimming, alone or in combination with at least one other active agent, in a cosmetic composition or for the preparation of a pharmaceutical and/or dermatological composition. Said peptides are intended, in particular, for treatment of cellulite and/or used to decrease, eliminate or prevent excess fat beneath the skin.
The study of the SIRT genes and the corresponding proteins has provided new therapeutic targets allowing for an intervention in the regulation of the energy metabolism of mammals. The SIRT proteins are part of the family of sirtuins, which are NAD+ dependent nuclear proteins responsible for deacetylation of histones (Rine J. et Al, Genetics, 1979). Several studies in organisms as diverse as C. Elegans or mammals have shown that there is a relation between energy input and life span and that the SIRT protein was responsible for this linkage. Thus, a reduction in calorie intake increases the life span of yeast (Tissenbaum et Al, Nature, 2001). Furthermore, recent studies have shown that SIRT1 causes mobilization of fat in the adipocytes (Picard F. et al, Nature, 2004). The SIRT proteins are currently considered to be potential therapeutic targets for treatment of dysfunctions or ailments connected with obesity, diabetes, or even hyperlipidemia, among others (WO 03/061681, US 2005/0164969).
The skin is the integument covering the entire surface of the body, made up of three distinct superimposed compartments: the epidermis (outer epithelium), the dermis (conjunctive tissue for support) and the hypodermis. The epidermis is a stratified epithelium constituting the external structure of the skin and providing its protective function. The dermis is a support tissue involved in the strength, the elasticity, and especially the tonicity of the skin and/or mucous membranes. Beneath the dermis is a layer of adipose tissues: the hypodermis.
The hypodermis is made up of a layer of white adipose tissue organized into lobules attached to the lower part of the dermis by rows of collagen fibers and elastic fibers. It is made up of large vacuolized cells, the adipocytes, almost entirely filled with triglycerides. These cells can rapidly change in volume as one loses or gains weight, and can measure from 40 to 120 μm in diameter, corresponding to a variation of 27 times in volume.
The hypodermal adipose tissue is the largest energy reservoir of the body. It is able to store lipids in the form of triglycerides by a process of lipogenesis, and then release them in the form of fatty acids and glycerol by a process of lipolysis. It is the equilibrium between these two metabolic pathways which governs adiposity. The lipid reserves of the body are constantly being renewed and have a close relation to the nutritional intake and energy needs of the body.
If a disequilibrium occurs in the body between the processes of lipogenesis and lipolysis, the volume and the number of adipocytes may increase; one speaks of adipocyte hypertrophy and hyperplasia. Excessive development of the adipose mass may then translate into modifications in the appearance of the skin, or even evolve into an overweight condition of the individual, possibly to the point of obesity.
Cellulite, considered to be unsightly, involves a hypertrophy of the adipose cells by overloading with triglycerides and a hyperviscosity of the ground substance (by polymerization of polysaccharides). These modifications impede exchanges between the cells and the mobility of the connective fibers (collagen, elastin and reticulin), which translates into water retention, slower venous and lymphatic circulation and loss of skin suppleness. The accentuation of the adipose tissue is localized, especially in women, in the area of the hips, thighs, buttocks, knees, and forearms. The skin takes on a quilted and padded appearance and in the most advanced stage an "orange peel" appearance, characterized by a succession of depressions caused by an excessive stretching of the connective segments and pulling of the epidermis toward the deep tissues.
At present, many research efforts have been carried out to find an effective way of fighting cellulite and excess fat in general. Many methods have been used, such as certain medical-surgical techniques like lipoplasty, lymph drainage, mesotherapy, ionophoresis techniques, etc. However, these techniques, while effective, are severe and restricting. Biological ways have been studied to act in a gentle and noninvasive manner on the mechanisms of formation of subcutaneous fat deposits.
Solutions have been proposed to intervene, in particular, in the metabolism of fatty acids. Cosmetic and pharmaceutical agents thus work on different levels to prevent the appearance of cellulite. For example, they will promote lipolysis, or rather inhibit lipogenesis, that is, reduce the formation of triglycerides.
The main objective of the present invention is to provide a new active ingredient for slimming. In fact, the inventors have succeeded in selecting particular substances having remarkable properties when applied to the skin.
Thus, by a first aspect, the object of the present invention is the use of an effective quantity of proteins of the SIRT family or peptide or polypeptide fragments of the SIRT family or biologically active derivatives of the latter, alone or in combination with at least one other active agent, to produce a localized slimming effect after being applied to the skin, and more particularly to reduce, eliminate or prevent fatty deposits under the skin.
According to one particularly advantage embodiment of the invention, the peptides belonging to the SIRT family are chosen among the peptides corresponding to the ID sequences N°:
(1)Gly Ala Gly Ile Ser Thr (2)Gly Ile Pro Asp Phe Arg Ser Pro (3)Gly Ile Pro Asp Phe Arg (4)Gly Ile Pro Asp Phe Arg Ser (5)Gly Leu Tyr Asp Asn Leu Glu (6)Thr Gln Asn Ile Asp Thr Leu
According to one especially preferred embodiment of the invention, the peptide fragment coming from the SIRT family has the ID sequence N° (5), that is, the sequence Gly Leu Tyr Asp Asn Leu Glu.
The invention also concerns the use of variant forms of these sequences and/or these fragments. The term "variant" denotes here a polypeptide or a peptide which differs, for example, from the sequence of a reference peptide while preserving its essential properties. Generally, the differences are limited such that the sequences of the reference peptide and those of the variant are quite similar and, in certain regions, identical.
Preferably, the variant forms are those which vary from the reference sequences by the substitution of chemically equivalent (or homologous) amino acids, that is, by the substitution of one residue for another one having the same characteristics. Thus, the classical substitutions are done between Ala, Val, Leu and Ile; between Ser and Thr; between the Asp and Glu acid residues; between Asp and Glu; between Asn and Gln; and between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. The term variant also denotes a peptide that differs, for example, from the sequence of a reference peptide while preserving its essential properties. Generally, the differences are limited such that the sequences of the reference peptide and those of the variant are quite similar and, in certain regions, identical. A variant peptide and a reference peptide can thus differ from the sequence of amino acids by one or more substitutions, additions, deletions in all combinations.
The fragments of SIRT proteins of polypeptide or peptide kind that are described in the present invention also include the use of all biologically active fragments, or one of their analogues or variants. By the phrase biologically active is also meant fragments having an in-vivo or in-vitro activity characteristic of the activity of the compound according to the invention.In the invention, the term "amino acid" refers here to any natural or nonnatural organic acid having the formula:—NHR—CR—C(O)—O—where each —R is selected independently between a hydrogen and an alkyl group having between 1 and 12 carbon atoms. Preferably, at least one —R group of each amino acid is a hydrogen. By the term "alkyl" is meant here a carbon chain which can be linear or branched, substituted (mono- or poly-) or nonsubstituted; saturated, monosaturated (a double or triple bond in the chain) or polyunsaturated (two or more double bonds, two or more triple bonds, one or more double bonds and one or more triple bonds in the chain).The term "peptide" denotes a chain of two or more amino acids joined together by peptide bonds or by modified peptide bonds; a polypeptide denotes a peptide of rather substantial size.By peptide is meant the natural or synthetic peptide of the invention as described above or at least one of its fragments, whether obtained by proteolysis or synthetically, or any natural or synthetic peptide whose sequence is totally or partially made up of the previously described peptide sequence.
In order to improve the resistance to degradation, it may be necessary to use a protected form of the peptide per the invention. The form of protection should obviously be a biologically compatible form and it should be compatible with a use in the field of cosmetics or pharmaceuticals.
Many forms of biologically compatible protection can be contemplated, and are well known to the practitioner, such as for example acylation or acetylation of the amino-terminal end, or amidation or esterification of the carboxy-terminal end. Thus, the invention concerns a use such as was defined above, characterized in that the peptide is in a protected form or not. Preferably, one uses a protection based on the acylation or acetylation of the amino-terminal end, or the amidation or esterification of the carboxy-terminal end, or both of these forms. The amino acid derivatives and the peptide derivatives also include amino acids and peptides linked together by a pseudo-peptide bond. By "pseudo-peptide bond" is meant all types of bonds able to replace the "classical" peptide bonds.
In the field of amino acids, the geometry of the molecules is such that they can in theory be present in the form of different optical isomers.
In fact, there exists one molecular conformation of the amino acid (AA) which deviates to the right the plane of polarization of light (dextrorotatory or D-aa conformation), and one molecular conformation of the amino acid (aa) which deviates to the left the plane of polarization of light (levorotatory or L-aa conformation). Nature has chosen for natural amino acids only the levorotatory conformation. Consequently, a peptide of natural origin will consist only of amino acids of type L-aa. However, chemical synthesis in the laboratory lets one prepare amino acids having the two possible conformations. Thus, starting with this base material, it is possible to incorporate both amino acids in the form of dextrorotatory or levorotatory optical isomers during the peptide synthesis. And so the amino acids constituting the peptide according to the invention can be in the L and D configuration; preferably, the amino acids are in the L form. The peptides according to the invention can thus be in L, D, or DL form.
The peptides in the present patent can be obtained either by classical chemical synthesis (in solid phase or in homogeneous liquid phase), or by enzymatic synthesis (Kullman et al., J. Biol. Chem. 1980, 225, 8234) from component amino acids or their derivatives.
The peptides of the invention can likewise be obtained by fermentation of a strain of bacteria, whether modified or not, by genetic engineering to produce the peptides of the above indicated sequence and their fragments, or by extraction of proteins of animal or vegetable origin, preferably of vegetable origin, followed by a controlled hydrolysis which liberates the peptide fragments of medium size and of small size, according to the invention.
Very many proteins found in plants are likely to contain these sequences within their structure. Guided hydrolysis makes it possible to release these peptide fragments. It is possible, but not necessary to realize the invention, to either extract the particular proteins at first and then hydrolyze them, or to perform the hydrolysis at first for a crude extract and then purify the peptide fragments. It is also possible to use certain hydrolyzed extracts without purifying the peptide fragments of the invention, yet still making sure of the presence of said fragments by suitable analytical means.
Other more simple or more complex procedures can be contemplated by the practitioner who is familiar with the field of synthesis, extraction and purification of proteins and peptides. Thus, the peptides of the invention can be of natural or synthetic origin. Preferably, according to the invention, the peptides are obtained by chemical synthesis.
In the composition per the invention, the peptides can be a mixture of peptide derivatives and/or be made up of amino acid derivatives.
The peptides of the invention, of the composition containing them, have a very effective action on the adipocytes. In fact, they help significantly decrease the quantity of triglycerides contained in the adipocytes of the hypodermis.
This phenomenon is probably due to a blocking of the phenomenon of lipogenesis, that is, blocking of the process of storage of triglycerides resulting in a hypertrophy of the adipocytes. A control of lipogenesis, that is, the reaction of synthesis of triglycerides in the adipocytes, will help prevent a hypertrophy of the adipocytes, as well as a succeeding hyperplasia. Thus, when in the course of adipocyte differentiation the quantity of triglycerides present in the vacuoles does not increase, the volume of the adipocytes and their number also will not increase. The skin gradually returns to its "normal" appearance. The cellulite tissue develops no further, and the orange peel effect is diminished. The unattractive appearance of the body is softened little by little.
The peptides of the invention or the composition containing them thus makes it possible, primarily because of their particular and pronounced activity on adipogenesis, to prevent the appearance of cellulite, and fight its worsening. If cellulite has become established, the peptides per the invention or the composition containing them will improve the appearance of the skin and, for example, attenuate the "orange peel" appearance. All of these properties make them particularly useful in cosmetic and dermatological preparation for slimming purposes.
It is quite obvious that the invention is addressed to mammals in general and more particularly human beings.
According to a second aspect of the invention, the aforesaid peptides can be used in or for the making of a topical-use composition intended for the treatment of cellulite and/or treatment of orange-peel skin. The peptides or the composition containing them are advantageously used to reduce, eliminate, or prevent excess fatty deposits beneath the skin.
According to one advantageous embodiment of the invention, the aforesaid peptides are first solubilized in one or more cosmetically or pharmaceutically acceptable solvents that are classically used by the practitioner, such as water, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclical polyols, vaseline, glycerol (glycerine, a vegetable oil, or any mixture of these solvents.
According to another advantageous embodiment of the invention, the aforesaid peptides are first solubilized in a cosmetic or pharmaceutical vector such as liposomes or adsorbed onto powderlike organic polymers, mineral supports such as talc and bentonite, and more generally solubilized in, or fixed to, any cosmetically or pharmaceutically acceptable vector.
The effective quantity of active ingredient corresponds to the quantity needed to obtain the desired result.
According to one advantageous embodiment of the invention, the aforesaid peptides are present, in the compositions of the invention, in a concentration between around 0.005 and 500 ppm (parts per million), and preferably in a concentration between around 0.1 and 50 ppm in weight as compared to the total weight of the final composition.
The composition containing the peptides per the invention can be a cosmetic or dermatological or pharmaceutical composition. Preferably, according to the invention, the composition is a cosmetic composition, since it is intended to improve the appearance and general performance of the skin of the person who uses it.
The composition per the invention is preferably a cosmetic and/or dermatological composition adapted for administration by topical skin route, containing a cosmetically or pharmaceutically acceptable medium.
These compositions could, in particular, be present in the form of an aqueous, water-alcohol, or oily solution, water-in-oil, or multiple emulsions; they can also be present in the form of suspensions, or powders, adapted to an application on the skin, the mucous membranes, the lips and/or the hair.
These compositions can be more or less fluid and have the appearance of a cream, a lotion, a serum, a pomade, a gel, a paste or a foam. They can also be present in solid form, such as a stick, or be applied to the skin in the form of an aerosol.
These compositions, moreover, contain any additive customarily used in the contemplated area of application, as well as the adjuvants needed for their formulation, such as solvents, thickeners, silicones, diluents, antioxidants, colorants, sun screens, self-tanning agents, pigments, ballasts, preservatives, perfumes, deodorants, cosmetic or pharmaceutical active agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.
For example, in said compositions the peptides per the invention can advantageously be combined with any other ingredient stimulating the exfoliation of the skin, inhibiting lipogenesis or stimulating lipolysis, such as derivatives of xanthine. One can mention, for example, caffeine, theophyllin, theobromin, acephyllin, nicotinate of xanthinol, diniprophyllin, diprophyllin, etamiphyllin and its derivatives, etophyllin, proxyphyllin, pentophyllin, propentophyllin, pyridophyllin and bamiphyllin.
In all these cases, the practitioner will make sure that these adjuvants, active or inactive, as well as their proportions are chosen in such a way as not to harm the advantageous properties sought from the composition per the invention. These adjuvants may, for example, correspond to 0.01 to 20% of the total weight of the composition.
If the composition of the invention is an emulsion, the fatty phase can represent from 5 to 80% by weight and preferably 5 to 50% by weight as compared to the total weight of the composition. The emulsifiers and coemulsifiers used in the composition will be chosen among those classically used in the field in question. For example, they can be used in a proportion ranging from 0.3 to 30% by weight, in relation to the total weight of the composition.
According to a third aspect of the invention, the aforesaid peptides can be used in or to make a pharmaceutical composition for topical use, intended for the treatment of cellulite and/or for the treatment of excess fat deposits beneath the skin.
According to a fourth aspect, the present invention concerns a method of cosmetic care to achieve a slimming effect. As well as a method of cosmetic care intended to reduce, eliminate and/or prevent excess fat deposits beneath the skin, and/or intended to fight cellulite and/or fight the orange-peel skin effect, consisting in applying, topically, to the zones of skin affected, an effective quantity of at least one of the aforesaid peptides.
The method of slimming cosmetic care also concerns the regular application of composition per the invention topically to the zones of the face or body being slimmed down, in particular the hips, the buttocks, the thighs, the stomach, the face.
The advantage of the present invention is that it can provide an effective topical treatment for adiposity while making use of "gentle" methods.
Other advantages and characteristics of the invention will appear better from a reading of the examples, given by way of illustration and not limitation.

Utilization of peptides as active ingredients for slimming


A composition comprising Transferrin binding proteins A and B is described (TbpA and TbpB). The composition is suitable for use in vaccines and for treatment of Gram negative bacterial infection, particularly meningococcal infection, demonstrating a broad spectrum of protection to a number of different bacterial pathogens. Also described are compositions comprising Tbps and other components, such as neisserial outer membrane vesicles and Cu,Zn-Superoxide dismutase. Methods for preparation of these compositions and their uses in vaccination against disease are further provided.

The present invention relates to a multicomponent vaccine and methods for preparing a multicomponent vaccine that confers protective immunity to a broad spectrum of infection by Gram negative pathogens. In particular the present invention relates to a multicomponent vaccine that provides both passive and active protective immunity to meningococcal disease.
Meningococcal meningitis is a major problem worldwide and in many countries incidence of infection is increasing. Neisseria meningitidis is the causative agent of the disease and is also responsible for meningococcal septicaemia, which is associated with rapid onset and high mortality, with around 22% of cases proving fatal.
At present, vaccines directed at providing protective immunity against meningococcal disease provide only limited protection because of the many different strains of N. meningitidis. Vaccines based upon the serogroup antigens, the capsular polysaccharides, offer only short lived protection against infection and do not protect against many strains commonly found in North America and Europe. A further drawback of these vaccines is that they provide low levels of protection for children under the age of 2 years, one of the most vulnerable groups that are commonly susceptible to infection.
The meningococcal transferrin receptor is made up of two types of component protein chain, Transferrin binding protein A (TbpA) and TbpB. The receptor complex is believed to be formed from a dimer of TbpA which associates with a single TbpB (Boulton et al. (1998)). Epitopes present in TbpA are known to be masked within the interior of the protein (Ala""Aldeen (1996)). Vaccines against meningococcal meningitis based on TbpB from one strain alone show some cross reactivity and there is evidence of a cross-reactive immune response in rabbits immunised with TbpB alone (Feirrerxc3x3s et al. (1998)).
Gomez et al (Vaccine Vol. 16 (1998) No. 17 pp. 1633-1633) describes a study on the effects of a number of different adjuvant compositions in vaccines comprising TbpA+TbpB complex. Immunization rates against meningococcal challenge were highest in compositions comprising TbpA+TbpB and RAS adjuvant.
Ala""Aldeen (J. Med. Microbiol. Vol. 44 (1996) pp. 237-237 provides a review of Tbps of N. meningitidis and their potential use as candidate vaccine antigens. Ala""Aldeen suggests that cross reactivity and broad spectrum protection is mainly associated with the TbpB subunit (page 240, third paragraph).
Gorringe et al (Vaccine Vol. 13 (1995) No. 13 pp. 1207-1207) describes the human antibody response to Tbps and evaluates their value as vaccine antigens by testing human sera for anti-Tbp antibodies. Gorringe et al does not describe the components of any such vaccine, but investigates the levels of antibody cross reactivity between purified TbpA and TbpB subunits and TbpA+B complex.
Boulton et al (Biochem. J. (1998) 334 pp. 269-273) describes the isolation and characterisation of TbpB from N. meningitidis and also postulates the make-up of the Tbp complex in vivo as a TbpA dimer plus TbpB monomer.
It would be of advantage, nevertheless, to provide a vaccine that gives a broader range of protective immunity to infection from a wider spectrum of strains of N. meningitidis. It would be of further advantage to provide a vaccine that confers protective immunity to infants as well as adults and whose protection is long term. It would also be of advantage to provide a vaccine that protects against sub-clinical infection, i.e. where symptoms of meningococcal infection are not immediately apparent that the infected individual may act as a carrier of the pathogen.
It is an object of the present invention to provide compositions containing Tbps, and vaccines based thereon, that meet or at least ameliorate the disadvantages in the art. In particular, it is an object of the invention to provide a vaccine composition that consistently and reliably induces protective immunity to meningococcal infection.
Accordingly, a first aspect of the present invention provides a composition comprising both transferrin binding proteins A (TbpA) and B (TbpB), suitably in a molar ratio of about 2:1 (TbpA:TbpB). In a preferred embodiment of the present invention the molar ratio of TbpA to TbpB is 2:1.
The composition may be combined with a pharmaceutically acceptable carrierxe2x80x94for example the adjuvant alum although any carrier suitable for oral, intravenous, subcutaneous, intraperitoneal or any other route of administration is suitablexe2x80x94to produce a pharmaceutical composition for treatment of meningococcal disease.
The present invention thus provides for a vaccine comprising both TbpA+B proteins, preferably with a molar ratio of between 1.8 and 2.2 molecules of TbpA to one molecule of TbpB, more preferably 2 molecules of TbpA to one of TbpB. This particular combination of components, surprisingly, can provide higher protective immunity to meningococcal infection, compared to vaccination with TbpB alone. In a specific embodiment of the invention, described in more detail below, a 1:1 combination of A:B is more protective against challenge than B alone. This is surprising as TbpA has previously been considered to be non-protective. The present results differ from this established view with some experiments (described in more detail below) showing that, when administered as a vaccine, TbpA is also able to provide protective immunity to meningococcal infection. However, the present results most strikingly demonstrate the consistent performance of vaccines that comprise both Tbps A and B compared to those comprising Tbp A or B alone. It is this lack of variability between compositions and the consistently high level of protection to infection induced in response to vaccination with Tbp A+B, that enables the compositions of the invention to demonstrate significant advantage over the vaccines of the prior art.
Transferrin binding proteins are known to be located on the outer membranes of a number of Gram negative bacteria such as N. meningitidis. Formulations of the composition of the present invention with conventional carriers or adjuvants provide a composition for treatment of infection by these bacteria.
It is an advantage that following administration of a composition according to the present invention antibodies may be raised against epitopes that consist of sequences from TbpA and TbpB in juxtaposition. Thus, the immune response obtainable using such a composition may be improved compared with that from prior art vaccine compositions which comprise only one component of the Tbp complex and in which the full range of potential Tbp epitopes are unavailable. It is a further option in the present invention for one Tbp subunit component of the TbpA+B complex to be from a first strain of N. meningitidis and another from a second strain different from the first. For example, the TbpA dimer is taken from the first strain and the TbpB is from the second. The TbpA and TbpB proteins may be selected independently from strains K454, H44/76 and B16B6. In all aspects of the invention the Tbps can be directly isolated from the bacterial source or can be produced by recombinant methods commonly known in the art. Combinations of proteins from other strains are also envisaged, and the combining of components from different strains of bacteria offers the potential for providing an individual with a broader spectrum of protection against meningococcal infection. It is further optional for a composition or vaccine of the invention to contain a mixture of A proteins from different strains or a mixture of B proteins from different strains, broadening further the potential spectrum of protection conferred by the invention. A still further option is for Tbps to be obtained from or derived from other bacteria, including N. gonorrhoeae, N. lactamica and Moraxella catarrhalis.
In the present invention, the term xe2x80x9ctransferrin binding proteinxe2x80x9d or xe2x80x9cTbpxe2x80x9d refers to a protein which either alone binds to transferrin or can be part of a complex of proteins that binds transferrin. The term also embraces fragments, variants and derivatives of such a protein provided that antibodies raised against the fragment, variant or derivative bind the protein. Thus, TbpA and TbpB either dissociated or associated into a complex are considered to be Tbp. Moreover, mutants, fusion proteins or fragments of either TbpA or B or other derivatives of the TbpA+B complex with a common antigenic identity are also considered to be represented by the term Tbp in the present invention.
A second aspect of the invention provides a composition comprising a complex of two TbpAs and one TbpB. The proteins are thus held together in the ratio seen in the native receptor. The individual proteins may be linked, for example, by hydrogen bonds or covalent bonds. In the latter case, each TbpA is covalently linked to the TbpA, either directly or indirectly. In a preferred embodiment, the complex of TbpA and TbpB assumes a native configuration.
A native TbpA+B complex may be isolated and purified from N. meningitidis. Alternatively, the invention also provides for synthesis of recombinant Tbp protein followed by assembly of the TbpA+B complex in vitro. The TbpA+B complex may be formed by admixture, or may be crosslinked by physical (e.g. UV radiation) or chemical methods known to the art, resulting in a combination of Tbps that will remain together and can not dissociate from each other. In a further example, a single chain recombinant protein comprising two TbpA sequences, preferably in the form of the TbpA dimer, is then covalently linked with TbpB protein to form a complete TbpA+B complex in vitro. Another example of the invention in use provides that TbpA and B are mutated so as to introduce cysteine residues that facilitate the formation of disuiphide bridges between the TbpA and TbpB subunits, so as to obtain a covalently bound complex.
In preparation of a recombinant protein TbpA and TbpB genes may also be truncated so that only those domains known to contribute to the antigenicity of the protein are incorporated in the Tbp complex.
In some compositions of the invention the TbpA+B complex is able to act as a transferrin receptor and binds to human transferrin. In other compositions of the invention the TbpA+B complex is non-functional in the sense that it does not bind transferrin, but it nevertheless provides an antigenic component that elicits an appropriate immune response.
A third aspect of the invention provides for a composition comprising a Tbp and N. meningitidis outer membrane vesicles. An advantage of this composition is that when administered to a vaccinee or patient it presents a different combination of N. meningitidis antigens, and particularly antigens that are in a formation substantially as present on the membrane of live infecting organisms. The combination offers the potential for a more effective protection against infection or a broader spectrum of protection than existing vaccines. Known methods of outer membrane vesicle isolation, such as by desoxycholate treatment, are suitable for preparation of compositions of the invention. In various preferred embodiments the Tbp is Tbp A, Tbp B or TbpA+B either in a native complex or in dissociated form.
The outer membrane vesicles may further be pretreated in vitro with Tbp so as to enrich the vesicle membrane with Tbp. By xe2x80x9cenrichxe2x80x9d and like terms, we refer to an outer membrane vesicle to which has been added Tbps so as to increase the concentration or density of Tbps in that vesicle. Preferably, the enrichment results in an outer membrane vesicle having an increased number of transferrin receptors located in the membrane, due to an increased concentration of TbpA and TbpB following their addition to the vesicle and their association into receptors or receptor-like structures. A particular advantage of such embodiments of the invention is that the Tbps, regarded as key antigenic components of the vaccine, are presented in a highly antigenic environment that closely mimics the environment in which transferrin receptors are presented on live, infecting bacteria.
As mentioned previously, the Tbp components of the composition need not be wild-type Tbps. They can be made recombinantly, and in so doing sequence alterations may be introduced. In one typical example of the invention, recombinant TbpB is modified so as to comprise a membrane binding domain. A preferred membrane spanning domain is a hydrophobic alpha helical region added to either the N or C terminus of the Tbp B protein. However, a membrane anchoring region need not only be an alpha helix, addition of a fatty acid or lipid chain would also facilitate membrane anchoring. In fact, wild type TbpB is believed to anchored to the bacterial outer membrane via such a lipid chain anchor. In a further example of the invention in use, the outer membrane vesicles are pretreated in vitro with membrane binding recombinant TbpB so as to enrich the vesicle membrane with TbpB. OMVs enriched with Tbps are additionally generated by inducing high levels of Tbp expression in N. meningitidis and then isolating OMVs via one of the methods described previously. This latter method is typically achieved by transforming the N. meningitidis host with a suitable expression vector into which has been inserted a gene or genes encoding the Tbps of choice. Suitable expression vectors for use in neisserial species include the plasmid pMGC10 (Nassif et al. (1991)).
It is preferred that the composition of the present invention comprises outer membrane vesicles and TbpA+B complexes isolated from a range of different strains of N. meningitidis. Other preferred compositions of the invention comprise other N. meningitidis proteins including surface antigens, periplasmic proteins, superoxide dismutase and glycoproteins.
The composition of the third aspect of the invention may instead of or in addition to outer membrane vesicles comprise one or more liposomes, each liposome including TbpA and/or TbpB preferably including TbpA and TbpB associated into a receptor or into a receptor-like complex. Thus a further means of presenting the transferrin receptor antigens is provided.
In a further embodiment of the third aspect of the invention, the composition comprises 22 kD antigen (Neisserial surface protein A (NspA)) as well as or instead of outer membrane vesicles. NspA and its preparation are described by Martin et al, 1997.
A fourth aspect of the invention provides for a vaccine comprising a composition of the invention as described above. A vaccine of the invention may also comprise antibodies to Tbp and thus provide a level of passive immunity to bacterial infection.
A fifth aspect of the invention provides for a method of manufacturing a composition that comprises combining TbpA, TbpB and N. meningitidis outer membrane vesicles with a pharmaceutically acceptable carrier. It is preferred that the molar ratio of TbpA to TbpB is about 2:1. The outer membrane vesicles can be pretreated in vitro with native TbpA+B so as to enrich the vesicle membrane with Tbp complex. However, the outer membrane vesicles may also be pretreated with other protein components so as to enrich them for these antigenic components also. The outer membrane vesicles may also be pretreated with antigenic proteins and proteoglycans from several different strains of N. meningitidis.
A further aspect of the invention provides for a composition comprising a Tbp and a Cu,Zn-Superoxide dismutase (Cu,Zn-SOD).
Cu,Zn-Superoxide Dismutase (Cu,Zn-SOD) is an metalloenzyme found in many prokaryotic and eukaryotic organisms. It catalyses the reduction of the superoxide radical anion, O2xe2x88x92, to hydrogen peroxide and molecular oxygen, thus playing an important role in the removal of cytotoxic free radicals from the organism. In bacteria Cu,Zn-SODs have been identified in the periplasm of a number of Gram negative species including N. meningitidis. The enzyme can exist as a dimer or a monomer, and accordingly in a preferred embodiment the present invention provides for a composition comprising a Tbp and a Cu,Zn-SOD of the dimeric type. As mentioned previously with regard to the use of the term xe2x80x9cTbpxe2x80x9d, the Cu,Zn-SOD of the present invention is also considered to encompass fragments, variants and derivatives of such a protein provided that antibodies raised against the fragment, variant or derivative bind the wild type Cu,Zn-SOD.
In examples of the invention in use, compositions are provided that comprise a Cu,Zn-SOD and either a TbpA, a TbpB or a TbpA+B complex. In the latter example it is preferred that the molar ratio of TbpA to Tbp B is between 1.8 and 2.2, with the most suitable compositions having a ratio of 2:1 (TbpA:TbpB). In further compositions of the invention the Tbps are from a different strain of N. meningitidis to that of the Cu,Zn-SOD, thus facilitating the formation of a broader spectrum of immune response to meningococcal infection.
The invention also provides for compositions wherein the Tbps and Cu,Zn-SOD are from different bacterial species, typically different Gram negative species. Such compositions thereby allow an even broader spectrum of immune response to be elicited when administered as a vaccine. Typical compositions comprise a neisserial TbpA+B complex as well as a monomeric Cu,Zn-SODxe2x80x94from E. coli for examplexe2x80x94and optionally a further dimeric Cu,Zn-SODxe2x80x94for example from Haemophilus parainfluenzae.
Other aspects of the invention provide for methods of manufacturing compositions that provide protective immunity to Gram negative bacterial infection, comprising combining a covalently linked complex of TbpA and TbpB with either or both of N. meningitidis outer membrane vesicles and a Cu,Zn-SOD, plus a pharmaceutically acceptable carrier.
A still further aspect of the invention provides for a composition comprising Tbps and outer membrane vesicles, wherein the outer membrane vesicles are from different strains of N. meningitidis. By analogy, the invention also provides for compositions comprising Tbps and a Cu,Zn-SOD, wherein the Tbps are from different strains of N. meningitidis.
A further aspect provides for use of Tbps A and B in the manufacture of a medicament for human vaccination. It is preferred that such a medicament is suitable for vaccination against meningococcal infection, although some compositions of the invention provide broad spectrum protection to infection from a wider range of bacterial pathogens.


An approach for predictively scoring test case results in real-time. Test case results associated with a test run are received by a software testing environment. Using predictive statistical models, test case results and attribute relationships are matched against model rules and test case history. A statistical correlation and confidence parameter provide the ability to generate test case relationships for predicting the outcome of other test cases in the test run. The test case relationships are transformed into scoring results and output for the further processing.

The present invention relates generally to the field of software testing, and more particularly to identifying a sequence of test cases in a test environment.
Software testing is an investigation conducted to provide stakeholders with information about the quality of the product or service under test. Software testing can also provide an objective, independent view of the software to allow the business to appreciate and understand the risks of software implementation. Test techniques include, but are not limited to, the process of executing a program or application with the intent of finding software bugs (errors or other defects).

This patent is subject to a terminal disclaimer.

Predictive model scoring to optimize test case order in real time

International Business Machines Corporation


A zero-offset wavefield synthesis workflow to calculate a synthesized zero-offset wavefield output without the commitment to an rms velocity field output to circumvent velocity uncertainty. Said zero-offset wavefield synthesis workflow comprises calculating a migration cube output. Rendering a demigration cube output from said migration cube output with a demigration cube calculation. Rendering said synthesized zero-offset wavefield output from said demigration cube output with a zero-offset wavefield synthesis procedure.

In areas with complex near-surface with irregular topography and structurally complex subsurface, there is much uncertainty in rms velocity estimation for prestack time migration. Whereas, interval velocity estimation for prestack depth migration is despairingly challenging. We often attribute the velocity uncertainty to various factors, including strong to severe lateral velocity variations, heterogeneity, anisotropy, mode conversion, and three-dimensional behavior of complex structures. Nevertheless, it is not easy to identify the cause of and account for the uncertainty as it often is a combination of the various factors. And the analyst struggles with much difficulty when estimating a velocity field whether it is for prestack time or depth migration.
Velocity uncertainty invariably gives rise to erroneously high or low migration velocities, which then causes two problems with prestack migration: (1) we fail to preserve reflector amplitudes, and (2) we also fail to position the reflectors correctly and focus diffractions to their apexes. We may choose to solve both problems simultaneously as we currently attempt to do with prestack migration workflows or we may choose to solve them one after the other as was done in the 1980s and 1990s by workflows that included Dip-Moveout (DMO) correction. The quality of image-gathers associated with prestack migration may not be adequate for velocity updating and thus may or may not warrant the simultaneous solution. In areas with irregular topography, complex near-surface, and complex subsurface, it may not. What then?
This disclosure proposes a workflow, applicable to both 2-D and 3-D seismic data, to solve the two problems with prestack time migration one after the other. The workflow is based on synthesis of a zero-offset wavefield to capture and preserve all reflections and diffractions, followed by zero-offset time migration.

Circumventing velocity uncertainty in imaging complex structures i-cube


A lottery ticket scraper constructed and arranged so as to scrap opaque coating from only portions of the lottery ticket, including means for supporting the lottery ticket, for aligning the lottery ticket and for scraping the lottery ticket are described.

Human history is cyclic. Civilizations rise from primitive cultures or previously decadent and crushed civilizations, often after a long period of darkness. As civilizations begin a birth, or the rebirth, moral values of the group, and ultimately of the individual rise and become dominate. The wealth of civilizations is often first enjoyed only by a small, select few, sometimes determined by heredity and sometimes by ability. The recognition of moral values, the integrity of the individual, the responsibility of each individual to look after his own needs and to care his own rises as a civilization grows toward maturity. Gradually, the oppressed and the less fortunate begin to partake of the feast of civilization and the conscience of society is turned toward taking care of those who are unable to care for themselves.
Those who have wealth and power are the first to gain education. Education, at first, is a means of enjoying the beauties of the earth, creating beauty, setting standards and values. As education progresses, it becomes more oriented toward occupations and trades and professions and means of earning livelihood. A strong desire for an educated population becomes a dominate drive of a maturing civilization, and the resources of the economy are focused toward educating and preparing those who will assume leadership in the future to assume that leadership is a responsible and ethical manner. As civilization reaches its crowning glory where all who have the drive and the will have the opportunity for education, all have the opportunity for work, and all have the opportunity for individual expression and growth and development, there creeps into civilization a growing proportion of hedonistic cultures whose goal in life is the profligate dissipation of life in sensualistic ritual and a subculture of torpid, shiftless and indolent individuals who live from the charity of others, either through the individual charity of those individuals who have wealth or through government taxation. This later subculture is generally at a very much lower economic status than the majority who have developed civilization to the point of individual responsibility and individual initiative and growth. As these two subcultures continue to grow and develop, they sap the industry and ingenuity of society to the point where a majority of society looks to the minority for its economic support, and a very small minority of society seek only for pleasure.
At this point, the importance of education continues to receive lip service, and the importance of the traditional social and economic values of individual integrity and responsibility continue to be the subject of rhetoric, but the values of society have so shifted that no segment of society desires to pay the price for quality education, but opiate their minds in the vast wastelands of gambling and television. Taxes have been increased to a confiscatory level, and society is unwilling to bear the burden of additional taxes. Demands upon government, as more individual payments are made and social benefits increase bring chaos and dependency. Deficit spending becomes the mode of financial planning, and alternatives to taxation are sought for.
There has existed in this society, from its beginning a small group of business men and others who thrive upon the weaknesses of individuals and of society, making their living through the pedaling of pornography, providing gambling parlors and casinos, and providing an endless train of mindless trivia which passes for entertainment. This group, seeing the opportunity to wedge itself into society through governmental power, persuades the people that they can waste their lives in indolence, attain wealth and solve society's problems through gambling sponsored by the government, bait their trap with a promise of better education for all. Society, thus, succumbs to its lowest elements, and government abandons its rule as a model of integrity and reliability. Thus, the state lottery is born.
With the state lottery comes one of the greatest artistic and wealth-creating inventions of all times, the Lottery Ticket! With the lottery ticket, however, comes a great problem which burdens society. Many lottery tickets include a backing of cardboard or other rigid stock material, printed indicia thereupon, and an opaque coating over some or all of the indicia. The lottery ticket, of this type, is utilized by the purchaser paying money into the state in the hope that by by scraping the ticket he will regain more money than he paid in. The promise is made is that someone will win millions of dollars. The state, however, is careful to minimize the odds against winning any substantial amount are several million to one. Not withstanding, these enormous odds against winning, the population flocks to the grocery store, the service station, the department store, the all-night liquor store, the drug store, and every other conceivable kind of merchandising operation wherein lottery sales have become a major source of activity. As the frantic lottery ticket purchaser grasps his ticket to instant wealth, he is confronted with a virtually insoluble dilemma--how to scrape the lottery ticket! First of all, one must have an object with a sharp edge. Coins are sometimes used, but these have a rounded edge and are most unsatisfactory. Pocket knives are sometimes used, but these tend to cut through the ticket, as well as fingers and thumbs, and destroy it. Nail files, credit card edges, razor blades and virtually every other conceivable kind of device with a sharp edge is used in the frantic race to scrape the opaque covering of the lottery ticket so as to become instantly rich. Most or all of these methods are only moderately satisfactory. One great hazard of most of these methods, lies in the fact that in some kinds of lottery tickets, if the number of the ticket, which is covered by the opaque covering, is disclosed by removing the opaque covering in that area, the lottery ticket is valid. Thus, sadly, the dream of instant wealth is cut asunder by a careless scrape!
The present invention solves this dilemma and frees oppressed society from the risk of destroying the validity of the lottery ticket, and provides a simple, efficient and effective means for scraping lottery tickets.


Method for the production of a laser-inscribable sheet of glass, wherein a laser-sensitive coating, comprising

The invention relates to the use of a laser-sensitive coating for the production of a laser-inscribable sheet of glass.
In the identification and labeling of products, control of manufacturing processes and for quality control, readable information is frequently applied to a component, including to glasses.
Hitherto, glasses, which can have a single-layer or multilayer structure, have been marked using labels, mechanical engraving or chemical etching methods. Labels are unsuitable for durable markings, and the two latter markings are frequently not used owing to the complex process.
The invention is based on the object of providing a laser-inscribable sheet of glass which is simple to manufacture, has high counterfeiting security and generally avoids or at least reduces the disadvantages of the prior art.
This object is achieved by using a laser-sensitive coating comprising
a) a base polymer and
b) a radiation-sensitive additive which exhibits a color change on laser irradiation.
The invention furthermore relates to a process for the production of a laser-inscribable multilayer glass.
Accordingly, the invention proposes the use of a laser-sensitive coating for the production of a laser-inscribable sheet of glass, where the laser-sensitive coating comprises a base polymer and a radiation-sensitive additive which exhibits a color change on laser irradiation. The coating system can be a colored or transparent coating system which exhibits decolorization on laser irradiation.
This laser-sensitive coating is applied to at least part of a sheet of glass and hardened. Suitable application methods are the known techniques, such as pouring and coating. The sheet of glass is subsequently inscribed by means of a laser.
The base polymer is preferably selected from a group consisting of unsaturated polyesters, epoxy, polyester and urethane acrylates, very particularly preferably an aliphatic polyurethane acrylate as also used for UV printing inks or EBC coatings in the furniture industry.
In addition, the base polymer is advantageously mixed with coloring pigments, light stabilizers, heat stabilizers or processing aids. The choice of said additives depends on which properties the laser-sensitive coating is to have.
The base polymer preferably contains the radiation-sensitive additive in a concentration in the range from 0.001% by weight to 3% by weight, based on the high-molecular-weight prepolymer, the radiation-sensitive additive being in particular a colored pigment or a metal salt, such as a copper salt or a titanium compound. Also suitable are white and colored pearlescent pigments based on mica and titanium dioxide or iron(III) oxide. Preference is given to copper hydroxide phosphate or Iriodin(copyright).
The additives are incorporated into the base polymer and are in finely divided form. The incorporation of the radiation-sensitive additive into the base polymer is carried out by known methods, for example by admixing the additive, in the form of a masterbatch, with the high-molecular-weight base polymer using an extruder, roll mill, mixer or grinder.
Preferred laser-sensitive pigments according to the invention are those which exhibit virtually no absorption in the visible wavelength region or, owing to the low concentration, give transparent or clear coating films.
In a particularly advantageous use, the laser-sensitive coating is applied between two sheets of glass and hardened, in which case the coating serves as bonding medium between the glass sheets.
The present invention also covers application of the coating to the outsides of laminated safety glass sheets, as used to obtain dirt-repellent or scratch-resistant sheets.
Another possible use of a laser-inscribable coating is for edge sealing of laminated safety glass sheets, in particular laminated vehicle windows, for example for the top edge of car windows which can be wound down. The base polymer can be applied to glass sheets by any conventional method, for example by scattering a powder or application of sheeting, followed by melting to give a strongly adherent film. It is likewise possible to apply aqueous emulsions.
Finally, the laser-sensitive coating can particularly advantageously be employed in a process for the production of a laser-inscribable multilayer glass, where the process comprises the following steps:
a) two glass sheets are bonded to one another in such a way that a cavity remains between them,
b) the cavity present between the glass sheets is filled with a coating material to such an extent that only a small partial region remains,
c) the laser-sensitive coating is introduced into the partial region,
d) the coating is then subjected to radiation-chemical curing, and
e) the multilayer glass is finally inscribed by means of a laser.
The inscription of the glass layers which are suitable in accordance with the invention is carried out using high-energy sources, such as lasers. Examples of such sources are solid-state lasers, such as neodymium-yttrium-aluminum-garnet lasers (Nd:YAG lasers), which emit at a wavelength of 1.06 xcexcm. Owing to the ready adjustability of their laser parameters, such as, for example, pulse frequency, lamp current and velocity of the laser beam, lasers can be used for precise matching to the needs of the materials to be inscribed.
In this case, a setting should be selected which means the greatest interaction for the laser-sensitive pigment, but does not give rise to expectations of any significant changes for the glass and the hardened coating film.
Incidence of the laser beam causes a color change with pronounced contrast at the irradiated points of the laser-sensitive film.
The process facilitates lifelong labeling of the glass sheet with barcodes, logos and with clear text, and in addition a marking which, in the case of multilayer glasses, is within the glass and consequently is extremely abrasion- and scratch-resistant. A labeling thus has equally high resistance as the glass itself.
The inscription is corrosion-resistant, dimensionally stable, deformation-free, and light-, heat- and weather-resistant. The novel coating material allows good legibility, high resolution and extremely small inscription sizes to be achieved.
In addition, the mechanical and physical properties of the glass are not, as in conventional processes, impaired by engraving or chemical etching. This should be particularly emphasized in the labeling of sensitive glasses, as used in windows in safety areas or in vehicles as windshields.
The simple and secure inscription of glass sheets, which can also take place, for example, on or in the glass sheet after installation in the vehicle and carries, for example, the vehicle number, can be used as an additional safeguard against theft.
However, the marking of the glasses with a brand is also readily possible, for example in the case of spectacle lenses.
In the laser-sensitive coating material according to the invention, the color change takes place through a direct color change of the additive itself or of the immediately adjacent vicinity through decomposition processes (carbonization) of the surrounding plastic matrix. The latter occurs in the particularly advantageous use of copper hydroxide phosphate. Foaming of the base polymer does not occur since only very few cracking gases, or none at all, are produced.
It is thus ensured that only very low emissions of cracking products, or none at all, occur during the inscription process.
Such cracking products can result, inter alia, in breaking of the bond in a multilayer glass sheet if care is not taken to divert such products beforehand.

Use of a laser-sensitive coating for the production of a laser-inscribable sheet of glass

Abstract: a) a base polymer and

Abstract: b) a radiation-sensitive additive which exhibits a color change on laser irradiation, is applied to at least part of a sheet of glass and hardened, and the sheet of glass is subsequently inscribed by means of a laser.